Method for making steroidal peracyl glycosides

ABSTRACT

Processes for the synthesis of tigogenin beta-O-cellobioside heptaalkanoate which is an intermediate for the known hypocholesterolemic agent tigogenin beta-cellobioside. The process comprises reacting α-cellobiosyl bromide heptaalkanoate and β-tigogenin in the presence of zinc fluoride or zinc cyanide under conditions capable of forming said tigogenyl β-O-cellobioside heptaalkanoate. The analogous preparations of hecogenin β-O-cellobioside heptaalkanoate, 11-ketotigogenin β-O-cellobioside heptaalkanoate, and diosgenin β-O-cellobioside heptaalkanoate are also disclosed. The process provides both high β-anomeric selectivity and high yields.

This application was filed under 35 U.S.C. §371 based on PCT/US92/08638,which was filed on Oct. 15, 1992 which is a continuation of U.S.application Ser. No. 07/797,574 which was filed on Nov. 25, 1991 and isnow abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to processes for the synthesis ofsteroidal glycosides, and particularly to the preparation of steroidalperacyl glycosides used as intermediates therein.

Tigogenin beta-O-cellobioside is a known compound having utility in thetreatment of hypercholesterolemia and atherosclerosis (Malinow, U.S.Pat. Nos. 4,602,003 and 4,602,005; Malinow et al., Steroids, vol. 48,pp. 197-211, 1986). Each patent disc;loses a different synthesis of thiscompound from alpha-D-cellobiose octaacetate; the first via the glycosylbromide heptaacetate which is coupled with tigogenin in the presence ofsilver carbonate, and finally hydrolyzed; and the second via directstannic chloride catalyzed coupling of the cellobiose octaacetate withtigogenin in methylene chloride, again followed by hydrolysis. InMalinow et al., reaction of cellobiose octaacetate with titaniumtetrabromide gave the cellobiosyl bromide heptaacetate, which wascoupled with tigogenin by means of mercuric cyanide, and thenhydrolyzed. All of these methods have serious drawbacks for producingbulk material to be used as a pharmaceutical drug. A desirable goal, metby the present invention, has been to devise synthetic methods whichavoid toxic and/or expensive reagents, and which cleanly produce thedesired tigogenin beta-O-cellobioside, avoiding tedious and expensivepurification steps.

Schmidt, Angew. Chem. Int. Ed. Engl., vol. 25, pp. 212-235 (1986) hasreviewed the synthesis and reactions of O-glycosyl trichloroacetimidatesformed by the reaction of sugars possessing a 1-hydroxy group (but withother hydroxy groups protected, e.g., by benzyl or acetyl) withtrichloroacetonitrile in the presence of a base. There is preferentialformation of the alpha-anomer when sodium hydride is used as base, andpreferential formation of the beta-anomer when the base is potassiumcarbonate. The alpha anomer of tetrabenzylglucosyl trichloroacetimidatewhen coupled with cholesterol gave anomerio mixtures which varied withcatalyst (p-toluenesulfonic acid or boron trifluoride etherate) andtemperature(-40° to +20° C.). On the other hand, both the alpha and betaanomers of tetraacetylglucosyl analog reportedly yield exclusivelybeta-anomeric products.

Thus, there has been a continuing search in this field of art forimproved methods of stereocontrolled syntheses of steroidal glycosides.

SUMMARY OF THE INVENTION

This invention is directed to a process for the synthesis of tigogeninβ-O, 11-ketotigogenin β-O, hecogenin β-O, or diosgenin β-O cellobiosideheptaalkanoate that provides greater β-anomeric selectivity andincreased yields. The process is particularly useful for preparingtigogenin β-O-cellobioside heptaalkanoate, which is an intermediate forthe known hypocholesterolemic agent tigogenin β-O-cellobioside. Theprocess comprises reacting α-cellobiosyl bromide heptaalkanoate andβ-tigogenin, 11-β-ketotigogenin, β-hecogenin or β-diosgenin in thepresence of zinc fluoride or zinc cyanide under conditions suitable forforming the tigogenin β-O-, 11-ketotigogenin β-O, hecogenin β-O-, ordiosgenin β-O-cellobioside heptaalkanoate.

Other features and advantages will be apparent from the specificationand claims.

DETAILED DESCRIPTION OF THE INVENTION

Preferably the metal salt used in the stereospecific reaction ofα-cellobiosyl bromide heptaalkanoate and β-tigogenin,11-keto-β-tigogenin, β-hecogenin or β-diosgenin is zinc fluoride or zinccyanide. It is especially preferred that the metal salt is zincfluoride. It is preferred that about 0.5 equivalents to about 4equivalents and especially preferred that about 1.5 equivalents to about2.25 equivalents metal salt is used.

It may also be preferred to conduct the zinc fluoride or zinccyanide-activated coupling in the presence of additional zinc salts suchas zinc halides (e.g., zinc bromide, zinc chloride, zinc iodide) orbasic salts of zinc (zinc oxide, zinc hydroxide, zinc hydroxy fluoride,zinc carbonate, etc.) to buffer or to activate the promoter (i.e., zincfluoride or zinc cyanide metal salt). Trialkyl tertiary amines (e.g.,diisopropylethyl amine, triethylamine, tributylamine), tetraalkylureas(e.g., tetramethyl urea, tetraethyl urea) or dialkylanilines (e.g.,diisopropyl aniline, dibutylaniline) are also useful reaction buffers.The above additives are generally used at 10-50%. mole equivalents ofthe promoters.

Although any of the alkanoate (C₁ -C₄) substituted alpha-cellobiosylbromides may be used it is preferred that acetate (i.e., C₁) is used.They may be prepared from conventional starting materials according tomethods described in K. Freudenberg and W. Nagai, Ann., 494,63 (1932)(e.g. Example 3). It is preferred that about 0.5 equivalents to about 3equivalents, and especially preferred that about 1 equivalent to about 2equivalents alkanoate (C₁ -C₄) substituted alpha-cellobiosyl bromidesare used.

Any reaction Inert solvent may be used. As used above and elsewhereherein, the expression "reaction-inert solvent" refers to a solventwhich does not react or decompose with starting materials, reagents,intermediates or products in a manner which adversely affects the yieldof the desired product. In general, the solvent can comprise a singleentity, or contain multiple components. Preferably the solvent is anon-protic reaction inert solvent and it is especially preferred thatthe solvent is acetonitrile because of the excellent stereoselectivityit provides. Other solvents include methylene chloride, ethyl acetateand nitromethane.

It is preferred that the reaction is acid catalyzed as this can increasethe selectivity of the β-cellobioside product over the α-cellobiosideanomeric product. Preferably mineral acids are used. Hydrobromic acidhas been shown to be particularly effective in increasing theβ-cellobioside product yield. Other preferred acids includehydrochloric, hydrofluoric and sulfuric acid. It is preferred that about0.05 equivalents to about 2 equivalents, and especially preferred thatabout 0.1 equivalents to about 0.5 equivalents add catalyst is used.

β-Tigogenin's preparation is described by Rubin in U.S. Pat. Nos.2,991,282 and 3,303,187, by B. Loken in U.S. Pat. No. 3,935,194 andCaglioti et al., Tetrahedron 19, 1127 (1963). Its structure is depictedbelow. ##STR1##

β-Hecogenin's preparation is described in a paper on SteroidalSapogenins by Russell E. Marker et al., in J. Amer. Chem. Soc., 69,2167-2211 (1947). Its structure is depicted below. ##STR2##

11-Keto-β-tigogenin switches the carbonyl group from the 12 position tothe 11 position of the structure depicted above. 11-Keto-β-tigogenin isprepared from hecogenin by the following procedure. According to theprocedure of Conforth, et al., (J. Chem. Soc., 1954, 907), hecogenin isacetylated, brominated, treated with sodium hydroxide and reduced withzinc to give the 12-hydroxy-11-keto analog. Then 12-hydroxy-11-ketoanalog is acetylated and reduced with calcium and ammonia to give11-ketotigogenin.

β-Diosgenin's preparation is described in "Diosgenin and Other SteroidalDrug Precursors" by Asoikar, L. V., Chadha, Y. R., and Rawat, P. S.,Council of Scientific and Industrial Research, New Delhi, India, 183pages, 1979 and also in T. Kawasaki et al., Chem., Pharm. Bull., Japan10 698 (1962). Its structure is depicted below. ##STR3##

Preferably about 1 equivalent to about 2 equivalents of the steroid isused. It is especially preferred that about 1 equivalent to about 1.5equivalents of the steroid is used.

Any environment or conditions (e.g., temperature, time, solvent)suitable for (i.e., capable of) forming the desired tigogenin,11-ketotigogenin, hecogenin- or diosgenin-beta-O-cellobiosideheptaalkanoate may be used. However, it is preferred that the reactionoccurs at a temperature of about 20° C. to about 100° C. and preferablyfrom about 50° C. to about 65° C. Below about 20° C. the reaction can beslow and above about 100° C. undesired side reactions (e.g.anomerization) can occur. This reaction is conveniently carried out atambient pressure however, pressures from about 0.5 to about 3atmospheres may be used.

Preferably the steroid, metal salt and solvent are heated to reflux andsufficient solvent is azeotropically distilled to remove substantiallyall the water. Then the cellobiosyl bromide heptaacetate is added to theabove mixture and heated for about 0.5 to about 6.0 hours, typicallyunder nitrogen. The desired compounds are then isolated by conventionalmethods.

For example, the glycosides may be precipitated from the crude filteredreaction mixture (e.g. acetonitrile product solution) by the addition ofabout 25% to 75% water and the remainder alcohol (e.g. methanol).Precipitation of the product from aqueous methanol/acetonitrile requiresless processing than an extractive isolation, and provides a product ofgreater purity.

The steroidal peracyl glycosides may be deacetylated by conventionalmethods such as treatment with triethylamine in methanol, basic anionexchange resins or sodium methoxide in methanol or methanol/THF solvents(e.g. Example 2 below). For example, the deacetylated product may beprepared by refluxing in methanol/THF using a non-catalytic amount ofsodium methoxide followed by conventional work-up. The excess methoxideis used to decompose the fluoro sugar, if any β-cellobiosyl fluorideheptaacetate is present, otherwise the deacetylation would be catalyticin sodium methoxide. The tigogenyl-β-O-cellobioside or analogs are thenisolated by conventional methods such as filtration.

Although the above process is designed to synthesize steroidalglycosides of the β configuration, the more thermodynamically stableα-anomers are accessible by acid-catalyzed isomerization of theβ-glycosides. For example, tigogenyl α-O-cellobioside heptaalkanoate canbe prepared from tigogenyl β-O-cellobioside heptaalkanoate by heatingthe β-glycoside in a methylene chloride solution containing hydrogenbromide.

The influence of reaction stoichiometry, temperature, solvents,molecular sieves, and vestigial hydrogen bromide on thestereoselectivity and yield of tigogenyl β-O-cellobioside heptaacetate(using the process of Example 1 ) are summarized in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Zinc Fluoride or Cyanide Activated                                            Glycosidic Couplings with Tigogenin                                           Equivalents                               β-Glycoside                    Glycosyl-Br                                                                          Activator                                                                             Tigogenin                                                                           Solvent  Sieves                                                                            Time/Temp.                                                                            Yield                               __________________________________________________________________________    2.00   ZnF.sub.2 (4.00)                                                                      1.0   CH.sub.3 CN                                                                            No  2.5 hrs/65° C.                                                                 79%                                 1.50   ZnF.sub.2 (3.00)                                                                      1.0   CH.sub.3 CN                                                                            No  2.5 hrs/65° C.                                                                 68%                                 0.50   ZnF.sub.2 (1.00)                                                                      1.0   CH.sub.3 CN                                                                            No  3.0 hrs/65° C.                                                                 32%                                 0.50   ZnF.sub.2 (1.00)                                                                      1.0   CH.sub.3 CN                                                                            No  1.5 hrs/65° C.                                                                 30%                                        Hbr(0.38)                                                              2.00   ZnF.sub.2 (4.00)                                                                      1.0   CH.sub.2 Cl.sub.2                                                                      No  3.0 hrs/43° C.                                                                 10%                                                                           25% (α-anomer)                2.00   ZnF.sub.2 (4.00)                                                                      1.0   Toluene  4Å                                                                            20 hrs/65° C.                                                                  41%                                 2.00   ZnF.sub.2 (4.00)                                                                      1.0   CH.sub.2 Cl.sub.2 /CH.sub.3 CN                                                         No  1.5 hrs/65° C.                                                                 64%                                                      (2/13)                                                   1.00   ZnF.sub.2 (2.00)                                                                      2.0   CH.sub.3 CN                                                                            No  1.0 hrs/65° C.                                                                 30%                                        Hbr(0.75)                                                              0.50   ZnF.sub.2 (0.50)                                                                      1.0   CH.sub.3 CN                                                                            No  22 hrs/50° C.                                                                  38%                                 2.00   ZnF.sub.2 (4.00)                                                                      1.0   CH.sub.3 CN                                                                            No  1.75 hrs/80° C.                                                                76%                                 0.50   ZnF.sub.2 (0.50)                                                                      1.0   CH.sub.3 CN                                                                            No  1.75 hrs/80° C.                                                                53%                                 1.25   ZnF.sub.2 (2.25)                                                                      1.0   CH.sub.3 CN                                                                            No  1.75 hrs/80° C.                                                                61%                                 2.00   Zn(CN).sub.2 (4.00)                                                                   1.0   CH.sub.3 CN                                                                            No  2.0 hrs/65° C.                                                                 63%                                 2.10   Zn(CN).sub.2 (5.60)                                                                   1.0   CH.sub.3 CN                                                                            No  3.0 hrs/65° C.                                                                 55%                                 1.50   Zn(CN).sub.2 (4.00)                                                                   1.0   CH.sub.3 CN                                                                            No  2.5 hrs/65° C.                                                                 45%                                 __________________________________________________________________________

The zinc fluoride-activated glycoside coupling was repeated withhecogenin and diosgenin in analogous processes to the β-tigogeninglycosidic coupling of Example 1 below. The results with these othersterols are summarized in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Zinc Fluoride-Mediated Glycosidic Couplings                                   with Hecogenin or Diosgenin                                                   Equivalents                        Yield                                      Glycosyl-Br                                                                          Activator                                                                          Sterol                                                                              Solvent                                                                            Sieves                                                                            Time/Temp.                                                                            β-Glycoside                           __________________________________________________________________________    2.00   2.25 Cholesterol                                                                         CH.sub.3 CN                                                                        No  2.25 hrs/65° C.                                                                63%                                                    (1.0)                                                             2.00   2.25 Hecogenin                                                                           CH.sub.3 CN                                                                        No  3.0 hrs/65° C.                                                                 72%                                                    (1.0)                                                             2.00   2.25 Diosgenin                                                                           CH.sub.3 CN                                                                        No  2.5 hrs/65° C.                                                                 65%                                                    (1.0)                                                             __________________________________________________________________________

This invention makes a significant advance in the field of steroidalglycosides by providing efficient methods of preparing steroidal peracylglycosides. The deacetylated end products are useful asantihypercholesterolemic agents.

It should be understood that the invention is not limited to theparticular embodiments shown and described herein, but that variouschanges and modifications may be made without departing from the spiritand scope of this novel concept as defined by the following claims.

EXAMPLE 1 Tigogenyl β-O-Cellobioside Heptaacetate

To a dry flask equipped with a mechanical stirrer, thermometer, anddistillation head were added β-tigogenin (4.16 g; 0.01 mole), anhydrouszinc fluoride (4.13 g; 0.04 mole), and 160 ml of dry acetonitrile. Theslurry was heated to reflux (85° C.) and 90 ml of distillates wasremoved overhead while 60 ml of fresh, dry acetonitrile was added to theslurry. The mixture was cooled to 25° C. and then a sample was removedfor a Karl Fisher determination (K.F.=0.02% H₂ O). α-Cellobiosyl bromideheptaacetate (14.00 g; 0.02 mole) was added to the flask, and then thestirred slurry was heated to 65° C. under a nitrogen atmosphere. Themixture was maintained at 65° C. for 2.5 hours when thin-layerchromatography¹ (tlc) showed that the reaction was complete. Thereaction was cooled to ambient temperature and 130 ml of methylenechloride was added. The thin slurry was filtered through Celite and thefiltrate (300 ml) was washed with a saturated sodium bicarbonatesolution (70 ml) followed by an aqueous wash (70 ml). After the organiclayer was dried over anhydrous sodium sulfate (20 grams) and filtered;the solution was then concentrated to 50 ml via a distillation atatmospheric pressure, Two hundred milliliters of 2B-ethanol was added tothe warm concentrate and the turbid solution was concentrated toapproximately 50 ml. The thin slurry was cooled to 25° C. and then itwas granulated for 90 minutes at room temperature. The crude product wasfiltered, the cake was washed with 25 ml of 2B-ethanol, and then driedat 40° C. in vacuo for 17 hours to give 10.8 grams of white crystallinesolids (m.p.=226°-231° C.).

The solids were dissolved in 25 ml of methylene chloride and then 75 mlof 2B-ethanol was added. The thin slurry was heated to reflux (760 mm)and 35 ml of distillate was removed overhead. The resulting slurry wascooled to room temperature and then was granulated for 90 minutes. Theβ-glycoside was filtered, and then dried at 40° C. in vacuo for 18 hoursto give 9.65 grams of a while crystalline solid (m.p.=229°-234° C.),Thin-layer chromatography¹ and high pressure liquid chromatography²(hplc) show that the product contains 77% (w/w) tigogenylβ-O-cellobioside heptaacetate and 15% (w/w) α-cellobiosyl fluorideheptaacetate. The α-cellobiosyl fluoride heptaacetate is most easilyremoved from the product during the deacetylation step.

EXAMPLE 2 Tigogenyl β-O-cellobioside

Crude tigogenyl β-O-cellobioside heptaacetate (50.0 g; 0.048 moles) wasdissolved in 250 ml of tetrahydrofuran and 250 ml of methanol whilemaintained under a nitrogen atmosphere. The hazy solution was filteredthrough a bed of Celite and then a solution of sodium methoxide (0.46 g;0.008 moles) in methanol (10 ml) was added to the filtrate. The solutionwas heated to reflux (60° C.) and maintained at reflux for 1.25 hoursgenerating a thick white slurry. A reaction aliquot was removed andanalyzed by thin-layer chromatography which indicated that the reactionwas complete. The slurry was concentrated by removing 200 ml ofdistillate and then 200 ml of water was added to the refluxing slurry.Another 200 ml of distillate was removed, and additional water (200 ml)was added. The slurry was cooled to ambient temperature and filtered.The product cake was washed with water (50 ml) and then pulled dry onthe filter. The water-wet cake was heated to reflux (65° C. in 600 mlsof THF and 92 mls of water). DARCO G-60 (1.53 grams) was added to thesolution, stirred for 15 minutes, and then the mixture was filteredthrough Celite. The solution was concentrated by removing 460 ml ofdistillates and 460 ml of methanol was then added. The methanol additionand concentration sequence was repeated twice again removing an addition800 mls of distillate and 800 mls of fresh methanol was added. Theresulting slurry was cooled to 20° C. and then granulated for one hour.The product was filtered, rinse with fresh methanol (50 ml), and thenthe wet cake was reslurried in 300 mls of fresh methanol (24° C.). Theproduct was filtered and then dried at 40° C. in vacuo overnight.Tigogenyl β-O-cellobioside (24.4 g; 0.036 moles) was isolated in 74%overall yield. Spectral and physical properties were identical to anauthentic sample.

EXAMPLE 3 α-D-Cellobiosyl Bromide Heptaacetate

α-D-Cellobiosyl bromide heptaacetate was prepared form α-D-cellobioseoctaacetate and hydrogen bromide in glacial acetic acid using a modifiedprocedure of Freudenberg and Nagari¹.

A 20% (w/w) hydrogen bromide solution (1.78.7 g; 0.44 mole of HBr) inglacial acetic acid was prepared by bubbling gaseous hydrogen bromideinto glacial acetic acid until a density of 1.212 was obtained. In aseparate dry reactor maintained under a nitrogen atmosphere,α-D-cellobiose octaacetate (50.0 g; 0.074 moles) was dissolved in 408 mlof methylene chloride. The HBr/HOAC solution was added to thedisaccharide solution to give a yellow solution. After the solution wasstirred for 2 hours at ˜17°-25° C., a small aliquot of solution wasremoved for a reaction completion assay. Once thin-layer chromatography²indicated that the reaction was complete, the solution was cooled to 10°C. and 0.5 liters of water was added. The mixture was stirred for 10minutes, the stirring was stopped, and the layers were allowed toseparate. The methylene chloride layer was decanted and then washed with7.5% w/w sodium bicarbonate solution (0.5 liters) followed by water (0.5liters). Finally, the methylene chloride solution was dried over 8 gramsof anhydrous magnesium sulfate and then filtered. The MgSO₄ hydrate cakewas washed with fifty milliliters of fresh methylene chloride, and thefiltrate and wash were combined. The methylene chloride solution wasconcentrated to approximately 0.15 liters by an atmospheric distillationand then cooled to ambient temperature. Diisopropyl ether (0.6 liters)was slowly added over 15 minutes with stirring to generate a thickslurry. The product was granulated for 1 hour at 25° C., filtered, andthen dried in vacuo at 40° C. for 4.5 hours. α-Cellobiosyl bromideheptaacetate (47.6 g; 92% yield) was obtained as a white crystallinesolid (m.p.=192°-194° C.) whose ¹ H NMR spectrum (CDCl₃) was consistentwith its structure.

EXAMPLE 4 11-Ketotigogenyl-β-O-Cellobioside Heptaacetate

To an appropriately equipped one liter, 3-necked round bottom flask wereadded acetonitrile (305 mls), 11-ketotigogenin (5.00 g; 0.011 moles),and rhombohedral, crystalline zinc fluoride (1.65 g; 0.016 moles). Theslurry was heated to reflux (80° C.) and then 100 ml of distillate wasremoved overhead. The slurry was cooled to room temperature, and then15.39 grams (0.022 moles) of α-cellobiosyl bromide heptaacetate wasadded. The reaction mixture was reheated to 60°-65° C. and thenmaintained at 60°-65° C. for 2 hours. A reaction sample was removed fora reaction completion assay. Thin-layer chromatography assay(EtOAc/hexanes 1.5:1) showed the complete disappearance of the glycosylbromide starting material so the reaction was cooled to 25° C. and 152ml of methylene chloride was added. After stirring for 10 minutes, themixture was filtered through Celite and the filter cake was washed with25 ml of CH₂ Cl₂. The combined reaction filtrate and wash were washedwith water (81 mls), saturated sodium bicarbonate solution (76 mls), andwater (137 ml). The organic layer was finally dried over 11 grams ofanhydrous magnesium sulfate. The MgSO₄ was filtered and washed with 16mls of fresh CH₂ Cl₂. The filtrate and wash were combined and thenconcentrated at reduced pressure to one fourth its original volume (300mls). 2B-Ethanol (250 ml) was added and the resulting solution wasconcentrated to one-half volume (170 mls). The slurry was cooled to20°-25° C. and then granulated for 1 hour. The white waxy solids werefiltered, washed with fresh 2B-ethanol (50 mls), and then dried in vacuoat 40° C. overnight. 11-ketotigogenyl-β-O-cellobioside heptaacetate (9.7grams; m.p.=205°-219° C.) was isolated in 84% overall yield.Chromatographic and spectral characterization were identical to anauthentic sample of 11-ketotigogenyl-β-O-cellobioside heptaacetate.Crude 11-ketotigogenyl-β-O-cellobioside could also be isolated in 64%yield by the following aqueous isolation sequence: The crude reactionmixture was diluted with additional fresh acetonitrile (50 ml) and thenfiltered through Celite. Methanol (290 mls) was added to the filtrateand the resulting solution was heated at reflux (65° C.) for one hour.One hundred milliliters of deionized water was slowly added to therefluxing solution to give a hazy mixture. After 20 minutes at reflux(72° C.), the mixture was slowly cooled to more temperature and thengranulated at 23°-25° C. for 1 hour. The crude product was filtered andwashed with water. The filter cake was suspended in 2B-ethanol (75 ml)and the mixture was heated to reflux. The mixture was then cooled toambient temperature, filtered, and the solids were dried in vacuo at 40°C. overnight. 11-Ketotigogenyl-β-O-cellobioside heptaacetate wasisolated in 64% overall yield.

EXAMPLE 5 11-Ketotigogenyl-β-O-Cellobioside

11-Ketotigogenyl-β-O-cellobioside heptaacetate (9.7 g; 9.2 mmoles) wassuspended in 50 mls of methanol and 50 mls of tetrahydrofuran. Thesystem was purged with nitrogen and then a solution of sodium methoxide(0.10 g; 1.9 mmoles) in methanol (1 ml) was added. The solution washeated to reflux (61° C.) and then maintained at reflux for 1 hour.Thin-layer chromatography (CH₂ Cl₂ /methanol 4:1) of the resultingslurry showed that the reaction was complete. The tetrahydrofuran wasremoved from the reaction by an atmospheric distillation and eventuallydisplacement with methanol (220 mls). A total of 180 ml of distillatewas collected. The slurry was cooled to 20°-25° C. and then granulatedovernight. The product was filtered, washed with methanol (2×20 mls) andthen the wet cake was reslurried (20 hours) in 100 ml of deionizedwater. After filtration and drying in vacuo at 25° C. overnight, 4.7grams of 11-ketotigogenyl-β-O-cellobioside was isolated in 65.5% overallyield. The product was homogenous by tlc and analyticalcharacterizations was consistent with the product's structure.

EXAMPLE 6 In Situ Hydrobromic Acid Generation

α-Cellobiosyl bromide heptaacetate (5.00 g; 7.15 mmole) and 50 ml ofacetonitrile were added to a 75 ml round bottom flask which was equippedwith a mechanical stirrer, thermometer, and vacuum distillation head.The system was purged with nitrogen and then the pressure was reduced toapproximately 425 mm Hg. The solution was heated to 56°-58° C. andapproximately 13 ml of distillate was removed overhead. The solution wascooled to room temperature and the vacuum was slowly released. Methylenechloride (37 ml) was added to the glycosyl bromide solution and then thesolution was extracted with water (2×25 ml). The aqueous phases werecombined and then titrated to a phenolphthalein endpoint using 0.1005 NNaOH solution.

The millequivalents of HBr acid contained in the α-cellobiosyl bromideheptaacetate solution before and after the azeotropic strip are reportedbelow. The azeotropic strip increase the tetratable acid approximately8-10 fold depending upon the water content.

    ______________________________________                                        TITRATED ACIDS                                                                ______________________________________                                        Initial α-Cellobiosyl Bromide                                                                 2.1 milliequivalents                                    Heptaacetate Solution                                                         Azeotroped α-Cellobiosyl Bromide                                                             16.9 milliequivalents                                    Heptaacetate Solution                                                         ______________________________________                                    

When the azeotropic distillation was used in Example 1 to dry theglycosyl bromide solution and to increase its acid content, the reactiontime was decreased to 1.0 hour at 65° C. In addition, high yields andhigh β-anomeric selectivity were maintained.

EXAMPLE 7 Aqueous Isolation of Tigogenyl β-O-Cellobioside Heptaacetate

A zinc fluoride-mediated glycosidic coupling of β-tigogenin (0.915 mole)with α-cellobiosyl bromide heptaacetate (1.830 mole) in acetonitrile wasconducted according to the procedure of Example 1. Once the reaction wascomplete, the crude reaction mixture was worked-up by an aqueous methodto give high-quality tigogenyl β-O-cellobioside heptaacetate by thefollowing sequence.

The crude reaction mixture was filtered through Celite to affordapproximately 20 liters of a golden colored filtrate. The filtrate washeated (55°-60° C.) and concentrated at reduced pressure to about 10liters. The concentrated solution was cooled to 50° C. and 5.0 liters ofmethanol was added. Subsequently, 7.5 liters of deionized water wasslowly added over 30 minutes. A solid precipitated from solution onceabout 2 liters of water was charged. The mixture was heated to reflux(73° C.) and then maintained at reflux for 2 hours. The slurry wascooled to 25° C. and granulated overnight. The crude product was,filtered, washed with methanol (2×1.5 liters), and then dried in vacuoat 40° C. The crude solid (1.01 kg) was 95.5% pure by a hplc assay. Inaddition, the crude product contained only 1.3% of tigogenylα-O-cellobioside heptaacetate and no β-cellobiosyl fluorideheptaacetate.

Crude tigogenyl β-O-cellobioside heptaacetate (1.0 kg) was slurried in10.2 liters of 2B ethanol under nitrogen and then the mixture was heatedto reflux (78° C.). After the slurry was at reflux for 1.5 hours, themixture was cooled to 25° C. and then granulated for 12 hours. Theproduct was filtered, washed with fresh ethanol (2×300 ml), and finallydried in vacuo at 40° C. overnight. Tigogenyl β-O-cellobiosideheptaacetate (0.89 kg) was isolated in 74% overall yield from tigogenin.The white crystalline product was 98.9% pure by high pressure liquidchromatography and contained only 0.5% (w/w) of the isomeric α-anomer.

We claim:
 1. A process for the synthesis of tigogenin-,11-ketotigogenin-, hecogenin- or diosgenin-β-O-cellobiosideheptaalkanoate comprising:reacting α-cellobiosyl bromide heptaalkanoate##STR4## wherein R is C₁ -C₄ alkyl and a steroid selected from the groupconsisting of β-tigogenin, 11-keto-β-tigogenin, β-hecogenin andβ-diosgenin, in the presence of a metal salt selected from zinc fluorideand zinc cyanide under conditions capable of forming said tigogenin-,11-ketotigogenin-, hecogenin- or diosgenin-β-O-cellobiosideheptaalkanoate.
 2. The process as recited in claim 1 wherein the metalsalt is zinc fluoride, R is methyl and the reaction occurs in anon-protic reaction inert solvent.
 3. The process as recited in claim 2wherein the steroid is β-tigogenin or 11-keto-β-tigogenin.
 4. Theprocess as recited in claim 3 wherein said reaction occurs in theadditional presence of an acid catalyst.
 5. The process as recited inclaim 4 wherein the acid catalyst is hydrobromic or hydrofluoric acid.6. The process as recited in claim 5 wherein the reaction occurs in theadditional presence of zinc bromide, zinc chloride, zinc iodide, zinchydroxy fluoride, zinc oxide, zinc carbonate, zinc hydroxide, a trialkyltertiary amine, a tetraalkyl urea or a N,N-dialkylaniline.
 7. Theprocess as recited in claim 6 wherein said reaction occurs at about 20°C. to about 100° C., about 1 to about 2 equivalents 11-keto-β-tigogeninis used, about 0.5 to about 4 equivalents zinc fluoride is used, andabout 0.5 to about 3 equivalents α-cellobiosyl bromide heptaalkanoate isused.
 8. The process as recited in claim 7 wherein the solvent isacetonitrile and about 0.05 to about 2 equivalents of hydrobromic acidis used.
 9. The process as recited in claim 2 wherein the solvent isacetonitrile and additionally comprising the step of precipitating the1-O-steroidal peracyl-β-glycosides from the acetonitrile by the additionof about 25% to about 75% water and the remainder alcohol.
 10. Theprocess as recited in claim 2 additionally comprising the step ofdeacetylating the tigogenyl β-O-Cellobioside heptaalkanoate to formtigogenyl β-O-cellobioside.
 11. The process as recited in claim 10wherein the deacetylation occurs by treatment with sodium methoxide inmethanol.
 12. The process as recited in claim 2 wherein the steroid is11-ketotigogenin.